Part:BBa_K602005:Experience

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2011 Osaka

UV tolerance

Several D. radiodurans proteins related to DNA damage repair (PprI, PprM, PprA, RecA) were assayed for their ability to confer damage tolerance to host E. coli cells. Transformed, pre-cultured cells were induced with IPTG, plated and exposed to varying doses of UV radiation. Plates were then wrapped with aluminum foil to prevent further exposure and incubated for 16h. From colony counts, the survival percentages of irradiated samples relative to controls were calculated and used as an indicator of tolerance.

Both PprM and RecA increased tolerance to UV irradiation even in the absence of IPTG induction, indicating that low levels of expression were sufficient for the function of these parts. The tolerance effect of PprM was unexpected given its role as a modulator of the PprI-dependent response mechanism in D. radiodurans. This result indicates that PprM may regulate tolerance-related proteins endogenous to E. coli as well.

On the other hand, non-induced PprI and PprA actually decreased tolerance. While it is understandable that PprI, a global regulator of the DNA damage response, may not be able to confer tolerance in the absence of its usual downstream genes, it is not clear why the low level of expression expected in the absence of induction would result in decreased tolerance, although the metabolic burden caused by carrying the plasmid is suggested as one cause.

It is interesting to note that in all cases, tolerance is increased upon IPTG induction, even in the wild type.

Combination of tolerance parts

When composite parts combining two tolerance genes were constructed and tested (+IPTG induction), the combinations PprI+RecA and PprM+RecA showed significantly higher tolerance to UV irradiation. In particular PprI and RecA showed a high level of synergy, with the resultant tolerance being significantly higher than either gene alone. This agrees with the reported role of PprI as an inducer of RecA in D. radiodurans. On the other hand the high tolerance conferred by PprM+RecA may be the additive effect of the two component genes.

Mitomycin C tolerance

The D. radiodurans proteins were also tested for their ability to confer resistance to Mitomycin C, a potent drug that causes DNA crosslinking and double-strand breaks. Transformed, pre-cultured cells were induced with IPTG, exposed to 2µg/ml Mitomycin C for 2h then plated on agar and incubated for 16h. From colony counts, the survival percentages of irradiated samples relative to controls were calculated and used as an indicator of tolerance.

PprM and RecA were effective in conferring tolerance. Induced PprI and PprA parts did not significantly increase tolerance relative to the wild type. As UV irradiation and Mitomycin C exposure cause different types of DNA damage (UV causes pyridine dimerizations while Mitomycin C can cause double strand breaks) we conclude that PprM and RecA may have the ability to repair a wider range of DNA damage than PprI or PprA.

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2012 Osaka

To measure the DNA damage tolerance conferred by each gene, we measured the survival rate in the presence of some DNA damaging agents (such as Mitomycin C and Hydrogen peroxide). Transformants E. coli were exposed to the DNA damaging agents and then incubated for 2 hours. Then, the cells were spreaded on agar plates at different dilutions. The plates were wrapped with aluminum foil and incubated in the dark. Colony-forming units were scored after 16h incubation at 37°C.

Mitomycin C tolerance

PprI did not increase the tolerance alone, while PprI regulates the multiple DNA repair and protection pathways in response to radiation. As E. coli lacks downstream proteins of D. radiodurans, it is expected that PprI would not be able to confer tolerance on its own. PprA, which repairs blunt-ended breaks in DNA lesions, also did not confer tolerance. PprA may not protect against DNA damages induced by MitomycinC. On the other hand, PprM and RecA much increased the tolerance. PprM is known to be a modulator of the PprI-dependent pathway. However, our data indicated that it is protective for E. coli by itself.

We observed increases in viability when certain tolerance genes were combined. For example, PprI by itself did not increase tolerance but boosted the ability of RecA to confer tolerance. This agrees with the role of PprI as an inducer of RecA in radiodurans. The combination of PprM and RecA also showed increase in tolerance. Since PprM is thought to not regulate RecA, this results indicate that PprM may induce or modulate other, unknown proteins and some of these proteins may have homologs in E. coli that benefit from the presence of PprM.

Hydrogen peroxide tolerance

Each PprA and PprM alone decreased tolerance. It may be due to the cost of their expression in the stressful cellular environment at least. On the other hand, both PprI and RecA significantly increased tolerance, which agrees with the role of PprI as an enhancer of enzyme activities of catalases.

We observed increases in viability when certain tolerance genes were combined. PprA or PprM alone did not increase tolerance, the combination of PprI and other radiotolerance genes somehow improved the tolerance. This results confirms that PprI enhance the enzyme activities of catalases.